A.Preparationofcelllysates

1. Collectcells(confluentT-25)bytrypsinizationandspin.
2. Lysethepelletwith100µllysisbufferonicefor10min.For500,000cells,lysewith20µl.
3. Spinat14,000rpm(16,000g)inanEppendorfmicrofugefor10minat4°C.
4. Transferthesupernatanttoanewtubeanddiscardthepellet.
5. Determinetheproteinconcentration(Bradfordassay,A280,orBCA)(WeusetheBradfordassayfromBio-Rad.)
6. Takexµl(=yµgprotein)andmixwithxµlof2xsamplebuffer.
7. Boilfor5min.
8. CoolatRTfor5min.
9. FlashspintobringdowncondensationpriortoloADInggel.

B.Polyacrylamidegel(14.5cmx16.5cm)

1. Agaroseplug:1%agarosedissolvedin1xResolvinggelbuffer.(Imake50ml,keepmeltingitasIneedit,andre-addingwatertomaintainagaroseconc.)
2. Resolvinggel:24mlofa9%gel5.4ml40%acrylamide/bisacrylamide(29:1mix)3ml8xResolvinggelbuffer15.6mlwater12µlTEMED60µl20%ammoniumpersulfate
3. Stackinggel:8ml1ml40%acrylamide/bisacrylamide(29:1mix)2ml4xStackinggelbuffer5mlwater8µlTEMED21.6µl20%ammoniumpersulfate

C.Preparationofgel

1. Assembletheglassplatesandspacers(1.5mmthick).
2. Pouranagaroseplug(1-2mm).
3. Pourtherunninggeltoabout1cmbelowthewellsofthecomb(~20ml).
4. Sealwith1mlwater-saturated1-butanol.(Canstophereandleavegelasisovernightifyouwant.)
5. Whengelhasset,pouroffthebutanolandrinsewithdeionizedwater.
6. Pourthestackinggel(~5ml)andinsertthecombimmediately.
7. Whenthestackinggelhasset,placeingelrigandimmerseinbuffer.
8. Priortorunningthegel,flushthewellsoutthoroughlywithrunningbuffer.

D.Runningthegel

1. Afterflashspinningthesamples,loadintothewells.
2. BesuretouseMarkers.Weuse15µlBio-RadKaleidoscopePrestainedStandards#161-0324directly.
3. Runwithconstantcurrent(35-37mAwithvoltagesetat>300V).
4. Usualrunningtimeisabout2.5hr.

E.Usingprecastgels(ReadyGelsfromBio-Rad):

1. Assemblegelingelrig.
2. Prepareproteinsamples(10µgwillsuffice).
3. Use5µlofKaleidoscopestandard.
4. Runat200V(constantvoltage)for30min.

F.Preparationofmembrane

1. CutapieceofPVDFmembrane(MilliporeImmobion-P#IPVH00010).
2. Wetforabout30mininmethanolonarockeratroomtemp.
3. Removemethanolandadd1xBlottingbufferuntilreadytouse.

G.Membranetransfer

1. Assemble"sandwich"forBio-Rad"sTransblot.
2. Prewetthesponges,filterpapers(slightlybiggerthangel)in1xBlottingbuffer.Sponge-filterpaper-gel-membrane-filterpaper-sponge
3. Transferfor1hrat1ampat4°Conastirplate.Biggerproteinsmighttakelongertotransfer.FortheMini-Transblot,it"s100Vfor1hrwiththecoldpackandprechilledbuffer.
4. Whenfinished,immersemembraneinBlockingbufferandblockovernight.

H.Antibodiesanddetection

1. IncubatewithprimaryantibodydilutedinBlockingbufferfor60minatroomtemp.
2. Wash3x10minwith0.05%Tween20inPBS.
3. IncubatewithsecondaryantibodydilutedinBlockingbufferfor45minatroomtemp.
4. Wash3x10minwith0.05%Tween20inPBS.
5. DetectwithAmershamECLkit(RPN2106).

I.Strippingblot

1. Rinseblotoffwith0.05%Tween20inPBS.
2. PutblotintoKapakbagcuttoslightlybiggersizethanblot.
3. Addabout5to10mlStrippingbuffer.
4. RemoveasmuchairaspossIBLeandsealbag.
5. Immerseinto80°Cwaterbathandincubatefor20min.
6. Rinseblotoffwith0.05%Tween20inPBS.
7. Blockforabout1hrwith5%BSA/Tween20,orovernightwith3%BSA/Tween20.

Lysisbuffer:

0.15MNaCl

5mMEDTA,pH8

1%TritonX100

10mMTris-Cl,pH7.4

Justbeforeusingadd:1:10005MDTT

1:1000100mMPMSFinisopropanol

1:10005Me-aminocaproicacid

2xsamplebuffer:

130mMTris-Cl,pH8.0

20%(v/v)Glycerol

4.6%(w/v)SDS

0.02%Bromophenolblue

2%DTT

8xResolvinggelbuffer:100ml

0.8gSDS(addlast)

36.3gTrizmabase(=3M)

AdjustpHto8.8withconcentratedHCl

4xStackinggelbuffer:100ml

0.4gSDS(addlast)

6.05gTrizmabase(=0.5M)

AdjustpHto6.8

10xRunningbuffer:1L

30.3gTrizmabase(=0.25M)

144gGlycine(=1.92M)

10gSDS(=1%)--addlast

DonotadjustthepH!!

10xBlottingbuffer:1L

30.3gTrizmabase(=0.25M)

144gGlycine(=1.92M)

pHshouldbe8.3;donotadjust

Tomake2Lof1xBlottingbuffer:

400mlMethanol

200ml10xBlottingbuffer

1400mlwater

Blockingbuffer:0.5L

3%Bovineserumalbumin(FractionV)

MakeupinPBSandsterilefilter.

Thenadd0.05%Tween20.

Keepat4°Ctopreventbacterialcontamination.

Strippingbuffer:0.5L(sterilefiltersolutionandkeepat4°C)

0.2MGlycine,pH2.5

0.05%Tween20

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